G-protein-coupled receptors (GPCRs) relay the information provided by numerous hormones and neurotransmitters into intracellular signalling pathways, primarily through their coupling to heterotrimeric G proteins. Agonist stimulation of GPCRs also initiates their feedback desensitization, mostly mediated by GPCR kinases (GRKs) and β-arrestin (β-arr) proteins. Through their binding to agonist-occupied, GRK-phosphorylated receptors, β-arrs prevent further coupling to G proteins and promote GPCR endocytosis, thus leading to decreased signalling efficacy. In addition to their role in receptor desensitization, β-arrs can act as scaffolds, linking GPCRs to mitogen-activated protein kinase signalling pathways (Luttrell & Lefkowitz, 2002). When considering their interaction with β-arrs, GPCRs can be divided into two classes: class A receptors interact only transiently with β-arr and undergo efficient recycling when released from β-arr, whereas class B receptors stably associate with β-arr as a result of higher affinity, thus leading to the accumulation of intracellular receptor/β-arr complexes that prevent receptor recycling (Oakley et al, 2001). Solved crystal structures (Hirsch et al, 1999; Han et al, 2001), mutagenesis (Vishnivetskiy et al, 2002) and limited tryptic proteolysis studies (Gurevich & Benovic, 1993; Xiao et al, 2004) suggest that a conformational rearrangement of the β-arr molecule accompanies its interaction with the activated receptor. It has been proposed that known intramolecular interactions between the amino- and carboxy-terminal domains in the inactive state are modified in the active β-arr, suggesting that the domains move relative to each other on activation. In this process, the C-tail seems to be released, thus exposing its clathrin—and adaptin 2 (AP2)—binding sites and promoting interactions with the internalization machinery (Lin et al, 1999, 2002; Gurevich & Gurevich, 2003).
In addition to their interactions with GPCRs, β-arrs were recently found to interact with receptors of other classes including receptor tyrosine kinases, receptor serine and threonine kinases, as well as adaptor proteins such as Disheveled (Lefkowitz and Whalen 2004) indicating that βarrs could be sensing the activated states of a wide diversity of signalling molecules.
Despite the growing diversity in GPCR signalling mechanisms, definitions of drug efficacy are often linked to a scheme considering only the regulation of the classical G protein signalling. Within this framework, agonists are defined as drugs that stabilize an active receptor conformation that induces G protein activation, whereas inverse agonists favor an inactive receptor state that reduces spontaneous G protein signalling. The question arises as to whether this paradigm may be transferred to drug effects generated through the formation of metastable complexes involving scaffolding proteins such as β-arr. Because all studies describing β-arr-mediated MAPK signalling have concentrated on agonist drugs, little is known of how ligands that are commonly classified as inverse agonists may regulate the scaffold assembly that is crucial for such signalling.
In one study (Azzi et al, 2003), this question was addressed by assessing whether β2-adrenergic receptor (β2AR) and V2 vasopressin receptor (V2R) ligands with proven inverse efficacy on adenylyl cyclase (AC) activity could also regulate MAPK activation via receptor-mediated scaffold formation. It was found that, despite being inverse agonists in the AC pathway, the β2AR (ICI118551 and propranolol) and V2R (SR121463A) induced the recruitment of β-arr leading to the activation of the ERK cascade. Such observations indicate that the same drug acting on a unique receptor can have opposite efficacies depending on the signalling pathway considered.
The above study relied on the use of a bimolecular bioluminescence resonance energy transfer (BRET) assay. It was used to assess β-arrestin recruitment to β2AR or V2R. Fusion proteins consisting of GFP10 variant (GFP) covalently attached to the carboxyl tail of the receptor of interest (β2AR-GFP; V2R-GFP) were co-expressed with β-arrestin 2 fused at its carboxyl terminus to Rluc (β-arrestin-Rluc). After incubation of the transfected cells with different ligands, Deep Blue coelanterazine (Packard) was added and readings were collected using a modified top-count apparatus (BRETCount, Packard) that allows the sequential integration of the signals detected at 370-450 nm and 500-530 nm. The BRET signal was determined by calculating the ratio of the light emitted by the Receptor-GFP (500-530 nm) over the light emitted by the β-arrestin-2-Rluc (370-450 nm). The values were corrected by subtracting the background signal detected when the β-arrestin-2-Rluc construct was expressed alone.
While the results elicited from the above study were instructive, a necessary feature involved the construction of fusion proteins that included the receptors of interest. Ideally, a method could be devised in which receptor activation might be observed without first having to modify the receptors that are to be studied. Other features of such a method that would make it highly desirable for research and development endeavors include the following: (1) a high level of sensitivity; (2) an ability to provide quantitative results; (3) adaptability for use in large scale screening analyses; (4) an assay that requires the expression of a single recombinant constructs; and (5) a biosensor based on an intramolecular BRET signal.
There is a need, therefore, for a simpler method to measure receptor activity in living cells. The present invention seeks to meet this and related needs.